Temperature‐induced changes in dissociation constants (K_A) of agonists at cardiac β‐adrenoceptors determined by use of the irreversible antagonist Ro 03–7894

Abstract

1The positive inotropic responses of guinea-pig left atria and papillary muscles and positive chronotropic responses of right atria to sympathomimetic amines were examined at 380 and 30°C. 2 At the lower temperature, supersensitivity to orciprenaline and isoprenaline was exhibited as shifts of the dose-response curves to the left and significant reductions in EC50 values. 3 This supersensitivity could not be attributed to reduced metabolism since the experiments were performed in the presence of metanephrine (10-5M) and U-0521 (3',4'-dihydroxy-2-methylpropiophenone) (10-4M) as inhibitors of extraneuronal uptake and catechol-Omethyltransferase (COMT) respectively, and the agonists are not susceptible to neuronal uptake. 4 After incubation of the tissues with Ro 03-7894 (1-(5-chloracetylaminobenzfuran-2-yl)-2-isopropylaminoethanol), followed by its prolonged washout (> 2h), the maximum responses to isoprenaline and orciprenaline were depressed, confirming the apparently irreversible Iadrenoceptor antagonism. 5 Dissociation constants (KA) for isoprenaline and orciprenaline were determined from the equiactive concentrations obtained before (A) and after (A') incubation with Ro 03-7894, plotted as 1/A against 1/A' (KA = (slope -1)/intercept). 6 KA values were the same for orciprenaline in the three cardiac preparations and for isoprenaline in the atria. This applied at 380 and 30°C and indicates that the P-adrenoceptors mediating the inotropic and chronotropic responses of the guinea-pig heart do not differ. 7 The KA values of both agonists were, however, consistently and significantly lower at 300 than at 38°C, indicating an increase in affinity. 8 It is concluded that hypothermia-induced supersensitivity of cardiac tissue to sympathomimetic amines is associated with an increase in their affinity for the ,B-adrenoceptors.

Calculated and actual changes in β-adrenoceptor number associated with increases in rat and guinea-pig cardiac sensitivity

“…Dissociation constants for isoprenaline have been determined for cardiac pl-adrenoceptors by pharmacological and radioligand binding techniques. They are known to vary with the bathing temperature (Broadley & Williams 1983), the ionic composition of the medium and the presence of GTP (McPherson et a1 1985). Values ranging from about 25 nM for both binding (Hawthorn & Broadley 1982) and pharmacological determinations (Buckner et a1 1978;Broadley & Nicholson 1981;Broadley & Williams 1983) to 200 nM for binding (McPherson et a1 1985) have been calculated.…”

“…They are known to vary with the bathing temperature (Broadley & Williams 1983), the ionic composition of the medium and the presence of GTP (McPherson et a1 1985). Values ranging from about 25 nM for both binding (Hawthorn & Broadley 1982) and pharmacological determinations (Buckner et a1 1978;Broadley & Nicholson 1981;Broadley & Williams 1983) to 200 nM for binding (McPherson et a1 1985) have been calculated. If these values are substituted in the equation, the increase in receptor number required to account for the change in sensitivity due to catecholamine depletion by 6-hydroxydopamine ranges from 2.32-to 2.56-fold (132-156% increase).…”

A comparison of the properties of prenalterol and corwin at β1‐ and β2‐adrenoreceptors in vitro

“…It is possible that the ratios of the estimated affinities of prenalterol and convin for cardiac p1-adrenoreceptors, compared with vascular /3,-adrenoreceptors, have been distorted slightly by the fact that experiments using cardiac tissue were carried out at low temperature (32°C: see above). Under these conditions the affinities of agonists for P,-adrenoreceptors are increased slightly (Broadley & Williams, 1983), although antagonist affinity is not affected (Reinhardt, Wagner & Schumann, 1972). However, the influence of the low temperature is unlikely to have had a major influence on the relative affinities of these partial agonists, prenalterol and convin, for /?,-compared with /?,-adrenoreceptors.…”

The classical competitive antagonism of phentolamine on smooth muscle preparations, investigated by two procedures

“…In brief, cooling of tissue changes their sensitivity to agonists (Foster, 1967; Harker & Van Houtte, 1989). At lower temperatures, the affinity of noradrenaline and isoproterenol to α ‐ and β ‐adrenoreceptors, respectively, was increased (Broadley & Williams; 1983; Raffa et al. , 1985).…”